Guidelines for Validation of Qualitative Real-Time PCR Methods for Molecular Diagnostic Identification of Probiotics

Abstract

Backgroud: Probiotics have been shown to benefit human health through several mechanisms, including their role in improving the health of our gastrointestinal tracts. The health benefits of probiotics are strain specific, and therefore it is critical to include the correct strains in probiotic products when claiming specific health benefits. Several studies have reported issues concerning the accuracy of labeling of commercial probiotic products, including inaccurate taxonomy, missing species, or undeclared species. Consequently, there is a growing need to develop and validate assays to reliably verify strain identity in commercial probiotic products. PCR-based methods are the most commonly used methods for food species ingredient diagnostics because they are simple, fast, sensitive, and can be validated. Objective: The aim of this paper is to set the guidelines for validating targeted qualitative real-time PCR assays to verify the presence of specific strains in a probiotic supplement. Methods and Results: Qualitative real-time PCR assays are validated to evaluate the assay performance in terms of specificity, sensitivity, repeatability, and reproducibility in detecting target strains. Conclusions and Highlights: Setting these guidelines will facilitate and streamline the validation process for qualitative real-time PCR-based assays for probiotic identity authentication in support of quality assurance systems.

Validation of a Triplex Quantitative Polymerase Chain Reaction Assay for Detection and Quantification of Traditional Protein Sources, Pisum sativum L. and Glycine max (L.) Merr., in Protein Powder Mixtures

“…Minimum performance thresholds for all triplex assay calibration were set to R 2 ≥ 0.98 (linearity) and 80–120% efficiency ( Bustin et al, 2009 ). For quantification purposes assay efficiency is recommended to fall within a narrower, 90–110% range ( Broeders et al, 2014 ). Material matrix type and DNA extraction methods can affect the dynamic range; thus, assay performance parameters are evaluated using target DNA extracted using two different methods, from two different matrices, at two different concentrations.…”

“…This involves proposed evaluation of applicability, analytical specificity, analytical sensitivity, efficiency, precision, dynamic range, repeatability, and reproducibility ( Newmaster et al, 2019 ). For quantitative assays that are intended to be used for more than detection, other considerations may be required, and acceptable ranges of performance parameters may be tighter, as to ensure sufficient precision ( Broeders et al, 2014 ).…”

“…An additional common recommendation in validation guideline literature for quantitative polymerase chain reaction (qPCR) assays is the evaluation of assay “practicability” ( Broeders et al, 2014 ; Newmaster et al, 2019 ). When designing any assay for eventual commercial applications, consideration must be given to cost of testing, required training of staff, affordability of equipment, and complexity of operations ( Newmaster et al, 2019 ).…”

Development of Real-Time PCR Assay to Specifically Detect 22 Bifidobacterium Species and Subspecies Using Comparative Genomics

“…Probiotics are living microorganisms that provide health benefits such as improving digestive health and preventing infectious diarrhea, irritable bowel syndrome, and inflammatory bowel disease of hosts (O'Callaghan and van Sinderen, 2016;Floch, 2018;Shehata et al, 2019). Health benefits of probiotics are species-or strain-specific.…”

“…Hence, differentiating Bifidobacterium subspecies is necessary. Furthermore, presenting correct species in probiotic products is critical for providing correct information to consumers and claiming health benefits of the product (Shehata et al, 2019). Recently, some studies have shown mislabeling issues such as absence of some species, inaccurate taxonomy information, and undeclared species (Lewis et al, 2016;Morovic et al, 2016) of commercial probiotic products.…”